A new form of directed cell migration, called mechanotaxis (or durotaxis), in which both speed and directional persistence are influenced by substrate mechanical properties, has been recently described in the literature. Exploiting mechanotaxis therapeutically, in conjunction with chemotaxis and haptotaxis, may represent a new approach to designing biomaterials that provide the appropriate combinations of chemical and mechanical cues to promote the selective migration of osteoblasts (or mesenchymal stem cells) from surrounding healthy tissue into small bony defects. Prior to pursuing craniofacial tissue engineering applications, additional basic science studies addressing the relevance of mechanotaxis for bone, combined with efforts to determine the mechanisms by which cells sense their local mechanical environment to initiate a program of cell motility, are required. This R03 application proposes a set of feasibility studies to determine if a pre-osteoblastic cell line (MC3T3-E1) will migrate via mechanotaxis on model substrates. Specifically, this proposal aims to: 1.) Fabricate polyacrylamide hydrogels with pre-defined mechanical compliance using a photopolymerization process, and to subsequently functionalize these hydrogels with extracellular matrix (ECM) proteins to support MC3T3-E1 adhesion and spreading; 2.) Quantify cell migration speed and directional persistence of MC3T3-E1 cells on ECM-modified hydrogel substrates possessing uniform stiffness, sharp interfaces in stiffness, or gradients in stiffness; and 3.) Determine what role, if any, is played by the Rho GTPases in sensing substrate compliance and regulating the mechanotactic response. Successful completion of these aims will not only provide useful fundamental data regarding cell migration mechanisms, but may also guide future osteoconductive strategies that exploit this mechanotaxis paradigm. Such strategies will be explored in the applicant's future R21 and R01 proposals. [unreadable] [unreadable]